scholarly journals MicroRNA-195 Activates Hepatic Stellate Cells In Vitro by Targeting Smad7

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Li-Ying Song ◽  
Yu-Tao Ma ◽  
Cui-Fang Wu ◽  
Chun-Jiang Wang ◽  
Wei-Jin Fang ◽  
...  
Keyword(s):  
Hepatic Stellate Cells ◽  
Luciferase Activity ◽  
Stellate Cells ◽  
Luciferase Reporter ◽  
Reporter Plasmid ◽  
Smad Pathway ◽  
Qrt Pcr ◽  
Reporter Assays

Background and Aim. Aberrant activation of the TGF-β1/Smad pathway contributes to the activation of hepatic stellate cells (HSCs). MicroRNA-195 has been shown to regulate the activation of HSCs. The aim of this study was to investigate the role of miRNA-195 in HSCs activation. Methods. A liver fibrotic rat model induced by diethylnitrosamine was established. Dual luciferase reporter assays were performed to verify that Smad7 was the target of miRNA-195. The expression levels of miR-195, Smad7, and α-SMA in HSC-T6 transfected, respectively, with miR-195 mimic, inhibitor, or control were measured by qRT-PCR. The protein expression of Smad7 was detected by Western blot analysis. Results. Enhanced miR-195 and decreased Smad7 were observed in diethylnitrosamine-induced liver fibrotic rats (P<0.05). Dual luciferase reporter assays showed that the miR-195 mimic significantly suppressed the luciferase activity of a reporter plasmid carrying the binding site of miR-195 on the 3′UTR of Smad7 (P<0.05). The miR-195 mimics activated HSCs, further elevated miR-195 and α-SMA (P<0.01), and reduced the Smad7 level (P<0.05). The miR-195 inhibitors blocked the activation of HSCs, reduced the expression of miR-195 and α-SMA (P<0.01), and upregulated the expression of Smad7 (P<0.05). Conclusion. Collectively, we demonstrated that miRNA-195 activated HSCs by targeting Smad7.

scholarly journals Investigating the expression of autophagy genes during the in vitro activation of mouse hepatic stellate cells

2021 ◽  
Vol 63 (2) ◽  
pp. 21-26
Author(s):  
Minh Thanh Dang ◽  
◽  
Van Trinh Le ◽  
Quang Huy Do ◽  
Thi Hang Tran ◽  
...  
Keyword(s):  
Hepatic Stellate Cells ◽  
Lipid Droplets ◽  
Stellate Cells ◽  
Beclin 1 ◽  
Collagen I ◽  
Antifibrotic Therapy ◽  
Qrt Pcr ◽  
Oil Red O

This study aims to evaluate the expression level of autophagy genes during the activation of mouse hepatic stellate cells (HSCs) in vitro. The HSCs isolated from the mouse liver were cultured in vitro for 7 days. The activation of HSCs was evaluated by their morphology, the storage of lipid droplets by oil red O (ORO) staining, the expression of activation-related genes α-sma, collagen I, and quiescence-related gene lrat by qRT PCR and ICC staining (α-SMA). The expression of autophagy genes lc3b, beclin 1, atg12 were assessed by qRT PCR and ICC staining (LC3). The results showed that the isolated HSCs were activated after 3 days and 7 days of the culture in vitro. The activation was indicated by the morphological change of HSCs to myofibroblast-like cells, loss of lipid droplets, and increased expression of fibrotic genes α-sma and collagen I, decreased expression of lrat. Additionally, the expression of autophagy genes lc3b, beclin 1, and atg12 were significantly increased in the activated HSCs after the culture in vitrofor 3 and 7 days. This study contributes the preliminary results to further studies on the role of autophagy during the activation of HSCs, which may be exploited for the development of the antifibrotic therapy targeting autophagy.

scholarly journals Let-7a regulates EV secretion and mitochondrial oxidative phosphorylation by targeting SNAP23 in colorectal cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
You Dong Liu ◽  
Xiao Peng Zhuang ◽  
Dong Lan Cai ◽  
Can Cao ◽  
Qi Sheng Gu ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Metabolic Reprogramming ◽  
Luciferase Reporter ◽  
Mitochondrial Oxidative Phosphorylation ◽  
In Vivo Assays ◽  
Reporter Assays ◽  
Extracellular Mirna

Abstract Background MicroRNAs (miRNAs) are abundant in tumor-derived extracellular vesicles (EVs) and the functions of extracellular miRNA to recipient cells have been extensively studied with tumorigenesis. However, the role of miRNA in EV secretion from cancer cells remains unknown. Methods qPCR and bioinformatics analysis were applied for determining extracellular let-7a expression from CRC patient serum and cells. Nanosight particle tracking analysis was performed for investigating the effect of let-7a on EV secretion. Luciferase reporter assays was used for identifying targeted genes synaptosome-associated protein 23 (SNAP23). In vitro and in vivo assays were used for exploring the function of let-7a/SNAP23 axis in CRC progression. Bioenergetic assays were performed for investigating the role of let-7a/SNAP23 in cellular metabolic reprogramming. Results let-7a miRNA was elevated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We determined that let-7a could suppress EV secretion directly targeting SNAP23. In turn, SNAP23 promotes EV secretion of let-7a to downregulate the intracellular let-7a expression. In addition, we found a novel mechanism of let-7a/SNAP23 axis by regulating mitochondrial oxidative phosphorylation (OXPHOS) through Lin28a/SDHA signaling pathway. Conclusions Let-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS through SNAP23, resulting in the suppression of CRC progression, suggesting that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.

scholarly journals Chorionic Villous Mesenchymal Stem Cells Derived Exosomes Deliver miR-135b-5p to Trophoblasts, Promoting their Proliferation and Invasion by Targeting TXNIP via β-Catenin Pathway

2020 ◽  
Author(s):  
Yijing Chu ◽  
Yan Zhang ◽  
Guoqiang Gao ◽  
Jun Zhou ◽  
Yang Lv ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Tissue Injury ◽  
Luciferase Reporter ◽  
Rna Seq ◽  
Qrt Pcr ◽  
Reporter Assays ◽  
Pcr Assays ◽  
Proliferation And Invasion

Abstract Background: Human chorionic villous mesenchymal stem cells (CV-MSCs) are found to be a promising and effective treatment for tissue injury. Trophoblast dysfunction during pregnancies is significantly involved in the pathogenesis of preeclampsia (PE). This work was to understand how CV-MSCs regulated trophoblast function. Methods: In this study, we treated trophoblasts with CV-MSC-derived exosomes and RNA-seq analysis was used to understand the changes in trophoblasts. We examined the levels of TXNIP and β-catenin in trophoblasts by immunohistochemistry, western blot and qRT-PCR assays. Luciferase reporter assays and qRT-PCR assays were used to understand the role of miR135b-5p in the effects of CV-MSC-derived exosomes. The growth and invasion of trophoblasts was evaluated with the CCK-8 and transwell assays. Results: The treatment markedly enhanced the trophoblast proliferation and invasion. Furthermore, a significant decrease of TXNIP expression and inactivation of the β-catenin pathway in CV-MSCs exosomes-treated trophoblasts was observed. Consistent with these findings, TXNIP inhibition exhibited the same effect of promoting trophoblast proliferation and invasion as induced by CV-MSC-derived exosomes, also with the accompaniment of inactivation of β-catenin pathway. In addition, overexpression of TXNIP activated the β-catenin pathway in trophoblasts, and reduced the proliferation and invasion of trophoblasts. Importantly, miR135b-5p was found to be highly expressed in CV-MSC exosomes and interact with TXNIP. The miR-135b-5p overexpression significantly elevated the proliferation and invasion of trophoblasts, which could be attenuated by TXNIP overexpression. Conclusion: Our results suggest that TXNIP-dependent β-catenin pathway inactivation mediated by miR135b-5p which is delivered by CV-MSC-derived exosomes could promote the proliferation and invasion of trophoblasts.

scholarly journals Long noncoding RNA HOTAIR mediates the estrogen-induced metastasis of endometrial cancer cells via the miR-646/NPM1 axis

2018 ◽  
Vol 314 (6) ◽  
pp. C690-C701 ◽  
Author(s):  
Yun-xiao Zhou ◽  
Chuan Wang ◽  
Li-wei Mao ◽  
Yan-li Wang ◽  
Li-qun Xia ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Qrt Pcr ◽  
Reporter Assays ◽  
Ec Cells

LncRNA homeobox (HOX) transcript antisense intergenic RNA (HOTAIR) has been confirmed to be involved in the tumorigenic progression of endometrial carcinoma (EC). However, the molecular mechanisms of HOTAIR in EC are not fully elucidated. The expression of HOTAIR and miR-646 in human EC tissues was determined by qRT-PCR. The effect of miR-646 on EC cells was assessed by the cell viability, migration, and invasion using CCK-8 assays and transwell assays. RNA-binding protein immunoprecipitation assays and RNA pull-down assays were performed to explore the interaction between HOTAIR and miR-646. The regulation of miR-646 on nucleophosmin 1 (NPM1) was tested using luciferase reporter assays. MiR-646 expression was significantly decreased both in human EC tissues ( n = 23) and cell lines (Ishikawa and HEC-1-A) compared with the control. Moreover, miR-646 expression was negatively related to HOTAIR in human EC tissues ( n = 23). Our results also showed that miR-646 overexpression considerably attenuated the E2-promoted viability, migration, and invasion of Ishikawa and HEC-1-A cells in vitro. In addition, HOTAIR was confirmed to regulate the viability, migration, and invasion of EC cells through negative regulating miR-646. More importantly, we also demonstrated that NPM1 was the target of miR-646, and HOTAIR promoted NPM1 expression through interacting with miR-646 in EC cells. Taken together, our findings presented that HOTAIR could regulate NPM1 via interacting with miR-646, thereby governing the viability, migration, and invasion of EC cells.

AK048794 maintains the mouse embryonic stem cell pluripotency by functioning as an miRNA sponge for miR-592

2016 ◽  
Vol 473 (20) ◽  
pp. 3639-3654 ◽  
Author(s):  
Yang Zhou ◽  
Qing-Song Dai ◽  
Shi-Chang Zhu ◽  
Yue-Hua Han ◽  
Hai-Long Han ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Luciferase Reporter ◽  
Stem Cell Pluripotency ◽  
Reporter Assays ◽  
Pluripotency Maintenance ◽  
Embryonic Stem Cell Pluripotency

MiR-592 has been identified as a neural-enriched microRNA, plays an important role in mNPCs differentiation, could induce astrogliogenesis differentiation arrest or/and enhance neurogenesis in vitro. Previous studies showed that long noncoding RNAs (lncRNAs) were involved in the neuronal development and activity. To investigate the role of miR-592 in neurogenesis, we described the expression profile of lncRNAs in miR-592 knockout mouse embryonic stem cells (mESCs) and the corresponding normal mESCs by microarray. By the microarray analysis and luciferase reporter assays, we demonstrated that lncRNA - AK048794, regulated by transcription factor GATA1, functioned as a competing endogenous RNA (ceRNA) for miR-592 and led to the de-repression of its endogenous target FAM91A1, which is involved in mESC pluripotency maintenance. Taken together, these observations imply that AK048794 modulated the expression of multiple genes involved in mESC pluripotency maintenance by acting as a ceRNA for miR-592, which may build up the link between the regulatory miRNA network and mESC pluripotency.

scholarly journals lncRNA SNHG15 Induced by SOX12 Promotes the Tumorigenic Properties and Chemoresistance in Cervical Cancer via the miR-4735-3p/HIF1a Pathway

2022 ◽  
Vol 2022 ◽  
pp. 1-15
Author(s):  
Jiang Yang ◽  
Mei Yang ◽  
Huabing Lv ◽  
Min Zhou ◽  
Xiaogang Mao ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Molecular Mechanisms ◽  
Therapeutic Strategies ◽  
Luciferase Reporter ◽  
Major Health ◽  
Tumor Tissues ◽  
High Prevalence ◽  
Reporter Assays

Cervical cancer (CC) is one of the most common malignancies in females, with high prevalence and mortality globally. Despite advances in diagnosis and therapeutic strategies developed in recent years, CC is still a major health burden worldwide. The molecular mechanisms underlying the development of CC need to be understood. In this study, we aimed to demonstrate the role of lncRNA SNHG15 in CC progression. Using qRT-PCR, we determined that lncRNA SNHG15 is highly expressed in CC tumor tissues and cells. lncRNA SNHG15 knockdown also reduces the tumorigenic properties of CC in vitro, as determined using the MTT, EdU, flow cytometry, and transwell assays. Using bioinformatics analysis, RNA pull-down, ChIP, and luciferase reporter assays, we verified the molecular mechanisms of lncRNA SNHG15 in CC progression and found that lncRNA SNHG15 expression in CC cells is transcriptionally regulated by SOX12; moreover, lncRNA SNHG15 promotes CC progression via the miR-4735-3p/HIF1a axis. This study can provide a potential target for CC diagnosis or therapeutic strategies in the future.

scholarly journals LncRNA LINC01303 Promotes the Progression of Oral Squamous Cell Carcinomas via the miR-429/ZEB1/EMT Axis

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Bo Sun ◽  
Xianyu Zheng ◽  
Weilong Ye ◽  
Pengcheng Zhao ◽  
Guowu Ma
Keyword(s):  
Squamous Cell ◽  
Luciferase Reporter ◽  
Real Time Quantitative Pcr ◽  
Qrt Pcr ◽  
Cytoplasmic Rna ◽  
Transwell Invasion Assay ◽  
Dual Luciferase Reporter Assay

Objectives. The aim of this research was to uncover the biological role and mechanisms of LINC01303 in oral squamous cell carcinoma (OSCC). Materials and Methods. Real-time quantitative PCR (qRT-PCR) was used to determine LINC01303 expression in OSCC tissues. Subcellular distribution of LINC01303 was examined by nuclear/cytoplasmic RNA fractionation and FISH experiments. The role of LINC01303 in the growth of TSCCA and SCC-25 was examined by CCK-8 assay, colony formation, transwell invasion assay in vitro, and xenograft tumor experiment in vivo. Dual-luciferase reporter assay was used to verify the interaction between LINC01303 and miR-429. RNA pull‐down assay was used to discover miR-429‐interacted protein, which was further examined by qRT-PCR, western blot, and rescue experiments. Results. LINC01303 expression was higher in OSCC tissues compared with adjacent nontumor tissues. LINC01303 was found to be localized in the cytoplasm of OSCC cells. Knockdown of LINC01303 inhibited OSCC cell proliferation and invasion, whereas increasing the expression of LINC01303 showed the opposite effects. Furthermore, LINC01303 served as a miR-429 “sponge” and positively regulated ZEB1 expression. Moreover, LINC01303 promoted OSCC through miR-429/ZEB1 axis both in vivo and in vitro. Conclusions. LINC01303 plays an oncogenic role in OSCC and is a promising biomarker for OSCC patients.

scholarly journals Remodeling of hepatic stellate cells orchestrated the stroma-derived oxaliplatin-resistance through CCN3 paracrine in hepatocellular carcinoma

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xia Liao ◽  
Yang Bu ◽  
Fan Chang ◽  
Fengan Jia ◽  
Ge Song ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatic Stellate Cells ◽  
Critical Role ◽  
Stellate Cells ◽  
Clinical Samples ◽  
Pathway Activation ◽  
The Relationship

Abstract Background Hepatic stellate cells (HSCs) have a key role in fibrogenesis and in the filtrates of the hepatocellular carcinoma (HCC) stroma, in which they are remodeled and play a critical role in HCC progression. However, the precise role of HSCs trending, infiltration and paracrine in orchestrating the stroma-derived oxaliplatin-resistance in HCC is still vague. Methods The chemo-resistant models were established to explore the correlation between HSC cells and the condition of chemoresistance. The HCC clinical samples were collected to confirm this phenomenon. Then, the relationship between secretory CCN3 from oxaliplatin-resistant HCC and the infiltration of HSCs in associated HCC microenvironment was evaluated. Finally, the role and mechanism of HSCs remodeling in the orchestration of oxaliplatin-resistant HCC were explored. Results The increased infiltration of HSCs and collagen accumulation were found in the microenvironment of oxaliplatin-resistant HCC. The cDNA profiles of the oxaliplatin-resistant HCC was reanalyzed, and CCN3 was one of the significantly increased genes. In HCC clinical samples, the levels of CCN3 and α-SMA are positively correlated, and high expression of CCN3 and α-SMA are positively associated with malignant phenotype and poor prognosis. Then the enhanced abilities of migration and proliferation of HSCs, and elevation of the cytokines paracrine from HSCs relating to HCC malignancy were proved in vitro and in vivo, and which were related to CCN3-ERK signaling pathway activation. Conclusions HSCs remodeling are positively related to CCN3 paracrine in hepatocellular carcinoma, which orchestrated the stroma-derived resistance to chemotherapy in HCC.

scholarly journals Role of MiR-98 and Its Underlying Mechanisms in Systemic Lupus Erythematosus

2018 ◽  
Vol 45 (10) ◽  
pp. 1397-1405 ◽  
Author(s):  
Lin Xie ◽  
Jinhua Xu
Keyword(s):  
T Cells ◽  
Lupus Erythematosus ◽  
Cd4 T Cells ◽  
Opposite Effect ◽  
Luciferase Reporter ◽  
Qrt Pcr ◽  
Fas Mrna ◽  
Reporter Assays ◽  
Underlying Mechanisms

Objective.T-lymphocyte apoptosis plays a critical role in the pathogenesis of systemic lupus erythematosus (SLE). However, the underlying regulatory mechanisms of apoptosis in SLE remain unclear. The aim of this study was to explore the role of miR-98 in SLE and its underlying mechanisms.Methods.Western blotting and quantitative reverse transcription PCR (qRT-PCR) were used to analyze miR-98 and Fas expression. Luciferase reporter assays were performed to identify miR-98 targets. To modify miRNA levels, miR-98 mimics and inhibitor were transfected into cells. A lentiviral construct was used to overexpress the level of Fas in SLE CD4+ T cells. Gene and protein expression were determined by qRT-PCR and Western blotting. Apoptosis levels were evaluated by annexin V staining and flow cytometry.Results.Compared to those of healthy donors, miR-98 was downregulated in SLE CD4+ T cells, whereas Fas mRNA and protein expression were upregulated. Upregulation of miR-98 by mimic transfection protected Jurkat cells against Fas-mediated apoptosis at both mRNA and protein levels, while miR-98 inhibitor induced the completely opposite effect. Luciferase reporter assays demonstrated that miR-98 directly targeted Fas mRNA. Further, miR-98 inhibitor induced apoptosis in primary healthy CD4+ T cells through the Fas-caspase axis, while upregulation of miR-98 in SLE CD4+ T cells led to the opposite effect.Conclusion.The current study revealed that downregulation of miR-98 induces apoptosis by modulating the Fas-mediated apoptotic signaling pathway in SLE CD4+ T cells. These results suggest that miR-98 might serve as a potential target for SLE treatment.

scholarly journals CXCL6 promotes human hepatocyte proliferation through the CXCR1–NFκB pathway and inhibits collagen I secretion by hepatic stellate cells

2016 ◽  
Vol 94 (3) ◽  
pp. 229-235 ◽  
Author(s):  
Zhenghong Li ◽  
Qidi Zhang ◽  
Qingqing Zhang ◽  
Mingyi Xu ◽  
Ying Qu ◽  
...  
Keyword(s):  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Alanine Transaminase ◽  
Collagen I ◽  
Hepatocyte Proliferation ◽  
Aspartate Transaminase ◽  
Nfκb Pathway ◽  
L02 Cells

Hepatocyte proliferation and collagen I (COLI) secretion are important processes during liver regeneration. This study aimed to investigate the role of CXCL6 in hepatocyte proliferation and COLI secretion. Serum CXCL6 levels in patients with chronic hepatitis B (CHB) were examined and the effects of CXCL6 on the proliferation of L02 hepatocytes and the secretion of COLI from LX2 human hepatic stellate cells were evaluated. We found that serum CXCL6 levels increased gradually with disease progression of CHB, and there was positive correlation between serum CXCL6 level and alanine transaminase (ALT) and aspartate transaminase (AST). In vitro, CXCL6 promoted L02 proliferation but this was blocked upon CXCR1 knockdown. The level of phospho-IκBα was upregulated by CXCL6 but downregulated by CXCR1 siRNA in L02 cells. CXCL6 inhibited the secretion of COLI by LX2 cells, dependent on CXCR1 and CXCR2. Taken together, these data suggest that increased expression of CXCL6 during CHB could promote hepatocyte proliferation through the CXCR1–NFκB pathway and inhibit the secretion of COLI by hepatic stellate cells.

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